Knock-out

SpCas9 nickase (Cas9n D10A) contains a mutation allowing the endonuclease to create single-strand nicks, as opposed to DSBs. Pairing two opposite facing gRNA sequences with SpCas9 nickase is an efficient method of gene editing that prevents unwanted indels from forming.

The NHEJ repair pathway is the most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations. In most cases, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene. However, the strength of the knockout phenotype for a given mutant cell must be validated experimentally.